Tomato (Lycopersicon esculentum) is one of the major vegetables in human diets. Use of traditional breeding techniques has incorporated numerous beneficial traits into the tomato, including such traits as extended shelf-life, disease resistance, and sugar content. There is also a significant interest in the industry to develop tomatoes that express compounds that can impart health benefits to humans. These health benefits can be delivered by development of dietary supplements containing the extracted components and by production of these components directly in a food source for human consumption.
Flavonoids, especially flavonols such as quercetin and kaempferol, are believed to impart a number of health benefits if ingested in sufficient quantities. Studies have shown that flavonoids possess antioxidant and anticancer activities (Rice-Evans et al., Free Radical Research, 22:375-383, 1995). Additional health benefits include anti-platelet aggregation (Rice-Evans et al., Trends in Plant Science, 2:152-159, 1997), decreased blood viscosity, reduction in the severity, of inflammation and allergies (Cook et al., Nutritional Biochemistry 7:66-76, 1996), as well as other health beneficial effects (e.g. antiviral activity, anti-tuberculosis activity). Therefore, there is significant interest in developing plants accumulating high levels of flavonoids, especially flavonols.
The flavonol, quercetin, is biosynthesized from p-coumaryl-CoA (C6C3) and malonyl-CoA (C3). In a polyketide synthesis reaction (catalyzed by chalcone synthase) the phenylpropane unit acts as a starting compound for the successive addition of three acetate units (C2—from malonyl-CoA) to form, after cyclization, naringenin chalcone (C6C3C6). The flavone naringenin is generated by formation of the heterocycle C-ring, a reaction that is catalyzed by chalcone isomerase. Quercetin is then synthesized by successive oxidization reactions of naringenin that yield a double bond between C-2 and C-3 and hydroxylation of C-3 and C-3′. These oxidation reactions are carried out by flavanone-3-hydroxylase, flavonol synthase and flavonol-3′,5′-hydroxylase. The final products of the pathway, quercetin mono- and di-glycosides (isoquercitrin and rutin, respectively), are formed through the action of O-glycosyltransferases. The mono-glucoside of the flavonol kaempferol, which is found only in trace amounts in tomato, is synthesized in the same way but with the omission of the hydroxylation in the C-3′ position.
Studies have shown, and we have confirmed, that in domesticated tomato varieties the chalcone isomerase (CHI) gene is present but not expressed in the fruit peel. Additionally, none of the flavonol biosynthesis genes are expressed in the flesh of the tomato fruit. Both results clearly explain the observed lack of flavonol accumulation in domesticated tomato fruits. Thus, it has been assumed in the art that obtaining high flavonol tomatoes cannot be accomplished by using traditional breeding techniques, as CHI expression would remain blocked and the fruit flesh would continue to accumulate insignificant levels of flavonols. Modern recombination technology, or genetic modification, is now being used to selectively modify the flavonoid pathway to produce plants with elevated levels of flavonols. However, to date, enhanced expression of flavonols is substantially limited to the tomato peel. See, for example, Muir, S. R. et al. Overexpression of petunia chalcone isomerase in tomato results in fruit containing levels of flavonols Nature Biotechnology, 19: 470-474 (2001) (Muir et al. detected approximately 0.5 mg/kg fresh weight [0.0045 μg/mgdwt] of rutin in tomato flesh, which was at the limit of detection); WO 00/37652, Flavonoid Biosynthetic Enzyme; WO 99/37794, Methods and Composition for Modulating Flavonoid Content; WO 99/14351, Isoflavone Biosynthetic Enzymes, WO 00/53771, Genetic Manipulation of Isoflavonoids; and WO 00/04175 Methods and Composition for Modulating Flavonoid Content. Table 1 shows the flavonol accumulation in the fruit of transgenic and non-transgenic tomato plants as revealed by various research groups. The high levels of flavonol accumulation of the cherry tomato is due to the high proportion of peel to flesh in cherry tomatoes compared to standard sized tomatoes.
TABLE 1mg flavonolsμg/mg dryμg/mg dryμg/mg dryperTomatoweightweightweight57 g tomatoSourceVariety(total fruit)(flesh)(peel)(estimate)SYNGENTAZTV 8401890.13.01.2DomesticatedUNILEVER (CHI/Transgenic4,0001725Petunia)(used 2 mmMuir, S., Collins, G.,thick peel)Robinson, S., Hughes, S.,Nontrans-0.05Bovy, A., DeVos, R., vangenic(used 2 mmTunen, A., Verhoeyen, M.thick peel)(2001) NatureBiotechnology, 19:470-474Non-Transgenic - Crozier,Different.6A., Lean, M., McDonald,varietiesBlack, C. (1997) J. Agric.Food Chem, 45: 590-595Non-Transgenic - Crozier,Cherry2.3A., Lean, M., McDonald,tomatoesBlack, C. (1997) J. Agric.Food Chem, 45: 590-595Non-Transgenic -Different0.010.721.2Stewart, A., Bozonnet, S.varietiesMullen, W., Jenkins, G.,Lean, M., Crozier, A.(2000) J. Agric. FoodChem., 49: 2663-2669
The present invention recognizes that there is a significant need to produce domesticated tomato plants, using traditional breeding techniques, that accumulate significant amounts of flavonols in the peel, as well as in the flesh, of the tomato fruit.
There is also a need for a method to identify tomato germplasm that expresses the CHI gene in the peel of the tomato fruit.
There is a further need to identify tomato germplasm that expresses the flavonol biosynthetic pathway in the flesh of the tomato fruit.
An additional need in the art is to identify tomato germplasm that expresses the CHI gene in the peel of the tomato fruit and the flavonol biosynthetic pathway in the flesh of the tomato fruit.